PU6::klp-12_sgRNA vector (V000447)

Basic Vector Information

      • Vector Name:
      • PU6::klp-12_sgRNA
      • Antibiotic Resistance:
      • Ampicillin
      • Length:
      • 3481 bp
      • Type:
      • Worm Expression, CRISPR
      • Copy Number:
      • High Copy
      • Promoter:
      • C. elegans U6 snRNA pol III promoter
      • Cloning Method:
      • Restriction Enzyme
      • 5' Primer:
      • M13 (-40) forward
      • 3' Primer:
      • M13 (-48) reverse

PU6::klp-12_sgRNA vector Vector Map

PU6::klp-12_sgRNA3481 bp6001200180024003000pGEX 3'pRS-markerIn lacZ geneCeU6 promotergRNA scaffold3' CeU6M13 revlac operatorlac promoterCAP binding siteL4440oriAmpRAmpR promoterpBRforEco

Plasmid Resuspension Protocol:

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5.Store the plasmid at -20 ℃.

PU6::klp-12_sgRNA vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported                3481 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    PU6::klp-12_sgRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3481)
  AUTHORS   Friedland AE, Tzur YB, Esvelt KM, Colaiacovo MP, Church GM, Calarco 
            JA
  TITLE     Heritable genome editing in C. elegans via a CRISPR-Cas9 system.
  JOURNAL   Nat Methods. 2013 Jun 30. doi: 10.1038/nmeth.2532.
  PUBMED    23817069
REFERENCE   2  (bases 1 to 3481)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2013 Jun 30. doi: 10.1038/nmeth.2532."
FEATURES             Location/Qualifiers
     source          1..3481
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        402..901
                     /label=CeU6 promoter
                     /note="promoter for the C. elegans K09B11.12 U6 snRNA gene 
                     (Friedland et al., 2013)"
     misc_RNA        922..997
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    1005..1241
                     /label=3' CeU6
                     /note="region downstream of the C. elegans K09B11.12 U6 
                     snRNA gene"
     primer_bind     complement(1260..1276)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1260..1276)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1273..1295)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1284..1300
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(1308..1338)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1353..1374
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1491..1508)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1662..2250)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1742..1761)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2421..3281)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3044..3063
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3282..3386)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3454..3472
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"

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